Cell classification comes from the need to distinguish between different cell types or different phases of the same cell type according to cell characteristics. Distinct features of cellular morphologies and phenotypes are usually used to classify cells, which, however, do not work for cells having similar morphologies or phenotypes. Therefore, various biomarkers have been discovered as complementary approaches for cell classification. Within them, cellular mechanics is a promising biomarker for a large variety of applications.
Cellular mechanics is known to tightly relate to its biological functions and activities, such as proliferation, migration and gene expression (Bao and Suresh, Nature materials 2, 715-725(2003); Vogel and Sheetz, Nat Rev Mol Cell Biol. 7, 265-275 (2006)). For example, when a cell needs to move forward, the contractile force will be generated within the cell body (Stossel, Science 260, 1086-1094(1993)). As a response to the shear flow in the blood vessel, the human endothelial cells will significantly change the expression of shear-stress regulated gene (Mccormick et al., Proc. Natl. Acad. Sci. USA 98, 8955-8960 (2001)). Clinically it shows that highly malignant and metastatic cancer cells are responsible for more than 90% of cancer-related deaths (Wirtz et al., Nat. Rev. Cancer. 11, 512-522 (2011)). In addition to genetic and external environmental factors, biomechanics of the cancer cell is key determinant of its activities. It is consistently found from the various biophysical measurements that cancer cells are softer than normal and benign cells and that this cellular compliance correlates with an increased metastatic potential (Cross et al., Nat. Nanotechnology 2, 780-783 (2007); Guck et al., Biophys J. 88, 3689-3698 (2005)). This correlation is likely from the required optimal mechanical properties of the cancer cell to efficiently migrate through a 3D matrix and/or penetrate through an endothelium during metastasis. It thus strongly indicates that cellular stiffness could be an inherent biomarker to grade the cancer progression. Recently, the nucleus has emerged as a key interest in cell mechanobiology (Dahl et al., Circ. Res. 102, 1307-1318 (2008); Booth et al., Soft Matter 11, 6412-6418(2015); Friedl et al., Curr Opin Cell Biol. 23, 55-64(2011); Denais et al., Science 352, 353-358 (2016); Davidson et al., Cell Mol. Bioeng. 7, 293-306 (2014); Khatau et al., Sci. Rep. 2:488 (2012); Fruleux and Hawkins, J Phys Condens Matter 28, 363002 (2016); Fu et al., Lab Chip 12, 3774-3778 (2012)). Force-induced changes in nuclear shape could result in large-scale reorganization of genetic material within the nucleus (Dahl (2008)). Changes in nuclear stiffness are associated with diseases, such as Huntington-Gilford progeria syndrome (Booth (2015)). As the largest and stiffest organelle within a cell, the nucleus imposes a major physical barrier for cell migration (Friedl (2011)). For cells to migrate through tissue, the nucleus must undergo complex changes in position, shape, and stiffness in coordination with cytoskeletal dynamics (Denais (2016); Davidson (2014); Khatau (2012); Fruleux and Hawkins (2016); Fu (2012)). Thus, nuclear mechanical properties are of great interest because they are closely related to cellular functions and could provide useful biophysical signatures to classify cells.
In the past two decades, many efforts have been made to develop methods to probe mechanical properties (e.g., elasticity and viscosity) of cells, such as micropipette aspiration (Mitchison and Swann, J. exp. Biol. 31, 443-460 (1954)), optical tweezer (Tan et al., J Biomech Eng. 132, 044504(2010)), optical stretcher (Guck et al., Biophys J. 88, 3689-3698 (2005); Guck et al., Biophys J. 81, 767-784 (2001)), deformability cytometry (Otto et al., Nat Methods 12, 199-202 (2015)), atomic force microscopy (AFM) (Lulevich et al., V Proc Natl Acad Sci USA 107, 13872-13877(2010)), magnetic twisting cytometry (Wang et al., Science 260, 1124-1127 (1993)), and micro-rheology (Weihs et al., Biophys J. 91, 4296-4305 (2006)). However, most of existing methods only provide an average measurement of the mechanical properties of the whole cell and cannot directly assess nucleus. Currently, extracting mechanical properties of the nucleus required staining, along with additional information and assumptions about how forces are transmitted within a cell. For pristine mechanical information, the nucleus has to be isolated to allow AFM or micropipette measurement (Lammerding, Compr. Physiol. 1, 783-807 (2011)), which is not only invasive but also may bias the measurement because of the isolation of the nucleus from the natural environment.
Brillouin scattering is the phenomena of inelastic light scattering induced by acoustic phonon of a material. In order to separate the small (typically in the order of GHz) Brillouin frequency shift from elastically scattered light, high-resolution spectrometer such as a multi-pass scanning Fabry-Perot interferometer is usually used in conventional Brillouin spectroscopy (Lindsay S M, Burgess S and Shepherd I W, “Correction of Brillouin linewidths measured by multipass Fabry-Perot spectroscopy,” Appl. Opt. 16(5), 1404-1407 (1977)). Since the dynamics of acoustic phonon is directly linked to the viscoelastic properties of a material, mechanical information can be acquired by measuring the Brillouin frequency shift of the scattered light (Dil J G, “Brillouin scattering in condensed matter,” Rep. Prog. Phys. 45, 286-334 (1982)). However, this method is fairly slow due to the point-by-point scan of the spectrum. In addition, the throughput efficiency is limited to the finesse of the etalon. This bottleneck was overcome by using a virtually imaged phased array (VIPA) etalon that can generate large angular dispersion (Xiao S, Weiner A M and Lin C, “A Dispersion Law for Virtually Imaged Phased-Array Spectral Dispersers Based on Paraxial Wave Theory,” IEEE J. Quantum Electronics 40(4), 420-426 (2004)) and enables measuring all of the spectral components simultaneously by a CCD camera with high throughput. Using this type of spectrometer, laser-scanning confocal Brillouin microscopy in biological tissue was performed at low illumination power and integration time, where each point in the sample is illuminated sequentially. The Brillouin spectrum was analyzed to create Brillouin-based elasticity maps (Scarcelli G and Yun S H, “Confocal Brillouin microscopy for three-dimensional mechanical imaging,” Nature Photonics 2, 39-43 (2007); Girard M J A, Dupps W J, Baskaran M, Scarcelli G, Yun S H, Quigley H A, Sigal I A and Strouthidis N G, “Translating Ocular Biomechanics into Clinical Practice: Current State and Future Prospects,” Curr. Eye Res. 40(1), 1-18 (2015)).
Brillouin spectroscopy allows non-invasive measurement of mechanical properties by measuring the frequency spectrum of acoustically-induced light scattering within a sample. Brillouin microscopy at sub-micron resolutions enabled the measurement of cell physico-chemical properties (Scarcelli G, Polacheck W J, Nia H T, Patel K, Grodzinsky A J, Kamm R D, Yun S H, “Noncontact three-dimensional mapping of intracellular hydromechanical properties by Brillouin microscopy”, Nat Methods. 12, 1132-1134 (2015)). In fact, cell mechanical properties critically regulate many cellular functions, e.g. proliferation, migration, gene expression as well as system-level behaviors, e.g. tissue morphogenesis, metastasis, and angiogenesis. As a result, “mechanical phenotyping”, i.e. the ability to classify cells based on their mechanical properties, has emerged as a powerful approach to characterize cell state and physiological/pathological conditions. For example, decreased cell stiffness has been shown to correlate with increased metastatic potential and thus it has been suggested as a novel label-free marker for tumor detection and staging (Swaminathan, V., et al. Mechanical Stiffness Grades Metastatic Potential in Patient Tumor Cells and in Cancer Cell Lines, Cancer Research 71, 5075-5080 (2011); Cross, S. E., Jin, Y. S., Rao, J. & Gimzewski, J. K. Nanomechanical analysis of cells from cancer patients. Nature Nanotechnology 2, 780-783 (2007)).
In related applications a system and method of label-free cytometry based on Brillouin light scattering has been provided which has successfully demonstrated its capability to directly probe the mechanical properties of the nucleus with submicron resolution (Zhang et al., Lab Chip 17, 663-670 (2017)). This method uses a light beam to sense the mechanical information of the sample by measuring the Brillouin frequency shift of the scattered light, and thus is intrinsically non-contact, non-invasive and label-free (Dil, Rep. Prog. Phys. 45, 286-334 (1982); Scarcelli and Yun, Nature Photonics 2, 39-43 (2007); Scarcelli et al., Nat Methods. 12, 1132-1134 (2015)).